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Disuse muscle atrophy is usually accompanied by changes in skeletal muscle structure, signaling, and contractile potential. Different models of muscle unloading can provide valuable information, but the protocols of experiments with complete immobilization are not physiologically representative of a sedentary lifestyle, which is highly prevalent among humans now. In the current study, we investigated the potential effects of restricted activity on the mechanical characteristics of rat postural (soleus) and locomotor (extensor digitorum longus, EDL) muscles. The restricted-activity rats were kept in small Plexiglas cages (17.0 × 9.6 × 13.0 cm) for 7 and 21 days. After this, soleus and EDL muscles were collected for ex vivo mechanical measurements and biochemical analysis. We demonstrated that while a 21-day movement restriction affected the weight of both muscles, in soleus muscle we observed a greater decrease. The maximum isometric force and passive tension in both muscles also significantly changed after 21 days of movement restriction, along with a decrease in the level of collagen 1 and 3 mRNA expression. Furthermore, the collagen content itself changed only in soleus after 7 and 21 days of movement restriction. With regard to cytoskeletal proteins, in our experiment we observed a significant decrease in telethonin in soleus, and a similar decrease in desmin and telethonin in EDL. We also observed a shift towards fast-type myosin heavy chain expression in soleus, but not in EDL. In summary, in this study we showed that movement restriction leads to profound specific changes in the mechanical properties of fast and slow skeletal muscles. Future studies may include evaluation of signaling mechanisms regulating the synthesis, degradation, and mRNA expression of the extracellular matrix and scaffold proteins of myofibers.

A giant multidomain protein of striated and smooth vertebrate muscles, titin, consists of tandems of immunoglobulin (Ig)- and fibronectin type III (FnIII)-like domains representing β-sandwiches, as well as of disordered segments. Chicken smooth muscles express several titin isoforms of ~500–1500 kDa. Using various structural-analysis methods, we investigated in vitro nonspecific amyloid aggregation of the high-molecular-weight isoform of chicken smooth-muscle titin (SMTHMW, ~1500 kDa). As confirmed by X-ray diffraction analysis, under near-physiological conditions, the protein formed amorphous amyloid aggregates with a quaternary cross-β structure within a relatively short time (~60 min). As shown by circular dichroism and Fourier-transform infrared spectroscopy, the quaternary cross-β structure—unlike other amyloidogenic proteins—formed without changes in the SMTHMW secondary structure. SMTHMW aggregates partially disaggregated upon increasing the ionic strength above the physiological level. Based on the data obtained, it is not the complete protein but its particular domains/segments that are likely involved in the formation of intermolecular interactions during SMTHMW amyloid aggregation. The discovered properties of titin position this protein as an object of interest for studying amyloid aggregation in vitro and expanding our views of the fundamentals of amyloidogenesis.

The changes in the content of the giant sarcomeric cytoskeletal proteins titin (3000–3700 kDa) and nebulin (770 kDa) in skeletal muscles (m. soleus, m. gastrocnemius), and titin in the left ventricular myocardium, as well as of the submembrane cytoskeletal protein dystrophin (427 kDa) in m. soleus and m. extensor digitorum longus (EDL), have been studied in the edible dormouse Glis glis during hibernation. The animals were divided into two experimental groups: “Summer activity” and “Hypothermia”. It was found that the development of atrophic changes in the skeletal muscles of hibernating animals is accompanied by a decrease in the dystrophin content. Specifically, the fluorescence intensity in skeletal muscle cross sections labeled with primary antibodies to dystrophin and Alexa Fluor® 488 conjugated secondary antibodies decreased in animals of the “Hypothermia” group by 2.7 times (p < 0.05) and 2.0 times (p < 0.05) in m. soleus and m. EDL, respectively. SDS electrophoresis of proteins in agarose-strengthened macroporous 2.2%-polyacrylamide gel revealed an insignificant decrease (by 15%, p ≤ 0.01) in the titin content compared to the myosin heavy chain content in m. gastrocnemius of animals of the “Hypothermia” group. The titin content in m. soleus and cardiac muscle, as well as the nebulin content in m. soleus and m. gastrocnemius, did not decrease during hibernation. These results are consistent with our previous data for other hibernators: long-tailed ground squirrel, brown and Himalayan black bears. It can be assumed that during evolution, hibernating animals developed the molecular mechanisms responsible for maintaining a stable level of giant sarcomeric cytoskeletal proteins during hibernation.

Changes in titin alternative splicing in the rat soleus after seven-day gravitational unloading (the hindlimb unloading model) were studied by long-fragment PCR and nanopore sequencing. An increase in the proportion of shorter titin mRNA transcripts was found in m. soleus of the hindlimb suspended rats, which may have led to the translation of shorter variants of the N2A isoform of this protein. In the nuclear fraction of m. soleus of control and hindlimb suspended rats, no significant differences were found in the content of Rbm20, an RNA-binding protein involved, as is shown earlier, in the regulation of titin alternative splicing in mammalian cardiac muscle. It can be assumed that Rbm20 is not involved in the regulation of titin alternative splicing in the rat soleus muscle.

In mammals, prolonged mechanical unloading results in a significant decrease in passive stiffness of postural muscles. The nature of this phenomenon remains unclear. The aim of the present study was to investigate possible causes for a reduction in rat soleus passive stiffness after 7 and 14 days of unloading (hindlimb suspension, HS). We hypothesized that HS-induced decrease in passive stiffness would be associated with calpain-dependent degradation of cytoskeletal proteins or a decrease in actomyosin interaction. Wistar rats were subjected to HS for 7 and 14 days with or without PD150606 (calpain inhibitor) treatment. Soleus muscles were subjected to biochemical studies and ex vivo measurements of passive tension with or without blebbistatin treatment (an inhibitor of actomyosin interactions). Passive tension of isolated soleus muscle significantly reduced after 7- and 14-day HS compared to control values. PD150606 treatment during 7- and 14-day HS induced an increase in alpha-actinin-2 and − 3, desmin contents compared to control, partly prevented a decrease in intact titin (T1) content and prevented a decrease in soleus passive tension. Incubation of soleus muscle with blebbistatin did not affect HS-induced reductions in specific passive tension in soleus muscle. Our study suggests that calpain-dependent breakdown of cytoskeletal proteins, but not a change in actomyosin interaction, significantly contributes to unloading-induced reductions in intrinsic passive stiffness of rat soleus muscle.

The effect of HDACs 4 and 5 on the level of atrophy, calpain-1 and titin content, and TTN gene expression in rat soleus after 7-day gravitational unloading (hindlimb suspension model) was studied. The development of atrophic changes induced by gravitational unloading in rat soleus was accompanied by an increase in the calpain-1 content, an increase in titin proteolysis, and a decrease in the mRNA content of the protein. Inhibition of HDACs 4 and 5 did not eliminate the development of unloading-induced atrophy but significantly prevented proteolysis of titin and the decrease in the TTN gene expression.

Objective: to elucidate the frequency and pathogenesis of myofascial pain syndrome (MFPS) in chronic nonspecific lower back pain (CNLBP) and to optimize the diagnosis and treatment of MFPS in CNLBP. Patients and methods. The investigation covered 121 patients with CNLBP. The patients' mean age was 42.1±10.5 years; the pain duration was 7.9±4.3 months. The possible causes of CNLBP were determined: these were facet joints (FJs); sacroiliac joints (SIJs); skeletal muscles with the development of MFPS; MFPS concurrent with FJs; MFPS concurrent with SIJs. Twenty patients had MFPS only (its mean duration was 5.3±2.2 months; the mean pain intensity scores were 6.5±1.1 on a numerical rating scale). Six patients underwent examinations of open biopsy specimens of the muscle straightening the spinal column; a comparison group consisted of 3 healthy women matched for age and gender. The patients were prescribed therapy with aceclofenac 200 mg/day in combination with tolperisone 450 mg/day and nondrug therapy (cognitive behavioral therapy and kinesio- and ergotherapy). When the treatment was insufficiently effective, ultrasonography of the muscle straightening the spinal column was additionally performed; a local anesthetic was injected into myofascial trigger points (MTPs). Results and discussion. MFPS was a cause of pain syndrome in 63 (52%) patients, while MFPS this was an isolated cause of pain in 20 (16.5%) cases and was concurrent with FJ osteoarthritis in 23 (19%), and with SIJ dysfunction in 20 (16.5%). Muscle ultrasonography in patients with MFPS revealed MTPs, whereas examinations of biopsy specimens of the muscle straightening the spinal column showed no evidence of necrosis, fibrosis, or inflammatory infiltration in the presence of transformation of the myosin phenotype, by increasing the proportion of rapidly fatigued type II muscle fibers. The results of sodium dodecyl sulfate (SDS) gel electrophoresis indicated a decrease in the content of titin and nebulin, the sarcomeric cytoskeletal proteins involved in maintaining muscle contractility. A two-week cycle of therapy with aceclofenac and tolperisone reversed pain syndrome in 5 (25%) of the 20 patients and reduced the intensity of back pain in 15 (75%), but the pain increased during physical exercise and impeded active rehabilitation. The additional administration of anesthetics into MTPs and the continuous intake of aceclofenac and tolperisone in combination with kinesiotherapy could relieve pain syndrome and enhance motor activity. Conclusion. More than half of the patients with CNLBP had MFPS only or concurrent with joint pathology (FS and FJs). The changes found in the back muscle biopsy specimens of patients with CNLBP are potentially reversible and can be reversed during kinesiotherapy.

The study was aimed at investigating the mechanisms and structures which determine mechanical properties of skeletal muscles under gravitational unloading and plantar mechanical stimulation (PMS). We hypothesized that PMS would increase NO production and prevent an unloading-induced reduction in skeletal muscle passive stiffness. Wistar rats were hindlimb suspended and subjected to a daily PMS and one group of stimulated animals was also treated with nitric oxide synthase (NOS) inhibitor (L-NAME). Animals received mechanical stimulation of the feet for 4 h a day throughout 7-day hindlimb suspension (HS) according to a scheme that mimics the normal walking of the animal. Seven-day HS led to a significant reduction in soleus muscle weight by 25%. However, PMS did not prevent the atrophic effect induced by HS. Gravitational unloading led to a significant decrease in maximum isometric force and passive stiffness by 38% and 31%, respectively. The use of PMS prevented a decrease in the maximum isometric strength of the soleus muscle. At the same time, the passive stiffness of the soleus in the PMS group significantly exceeded the control values by 40%. L-NAME (NOS inhibitor) administration attenuated the effect of PMS on passive stiffness and maximum force of the soleus muscle. The content of the studied cytoskeletal proteins (α-actinin-2, α-actinin-3, desmin, titin, nebulin) decreased after 7-day HS, but this decrease was successfully prevented by PMS in a NOS-dependent manner. We also observed significant decreases in mRNA expression levels of α-actinin-2, desmin, and titin after HS, which was prevented by PMS. The study also revealed a significant NOS-dependent effect of PMS on the content of collagen-1a, but not collagen-3a. Thus, PMS during mechanical unloading is able to maintain soleus muscle passive tension and force as well as mRNA transcription and protein contents of cytoskeletal proteins in a NOS-dependent manner.

—Here, we consider the problem of the activation of the complement system by amyloid aggregates, in particular, amyloid fibrils of the Aβ(1-40) and Aβ(1-42) peptides found in the brain of patients with Alzheimer’s disease . In 1992, based on the data on colocalization of amyloid inclusions with complement system proteins in the brains of patients with this disease, it was suggested that this system is directly activated by amyloids. This hypothesis has been supported by a number of subsequent publications. However, in this study, we did not find evidence to support this idea. The issue remains as to whether amyloids activate the complement system, and, if not, which mechanism is responsible for its activation. Here, we try to elucidate these issues.

In this paper, the property of the muscle titin protein to form in vitro specific amyloid-like aggregates is discussed. The main difference from the known amyloid aggregates is the formation of a quaternary structure that resembles cross-β, with no changes in the secondary structure. Based on the results obtained earlier, as well as the results of this study, we make assumptions about changes in the structure of titin that occur during the formation of amyloid-like aggregates. In particular, our X-ray diffraction data on the titin aggregates suggest that β-strands in the aggregates of this protein are not located perpendicular to the fibril axis, as described for other amyloid proteins, but in parallel. The distance between the β-sheets in the aggregates may vary, and the β-sheets themselves are not strictly oriented along one of the axes, which can lead to the appearance of a diffuse ring reflection of ~8-12 A. In this regard, the titin aggregates should not be called amyloid, but amyloid-like, with a quaternary structure that resembles cross-β. It cannot be excluded that the formation of this quaternary structure can also occur due to the partial unfolding of titin domains, followed by the interaction of open β-strands between neighboring domains and/or domains of neighboring molecules.

The giant muscle protein, titin, is the third most abundant protein in muscle (after myosin and actin). It was shown previously that smooth muscle titin (SMT) with a molecular mass of 500 kDa can form in vitro amorphous amyloid aggregates in two conditions: in solution of low ionic strength (0.15 M Glycine-KOH, pH 7.0) (SMT(Gly) aggregates) and in solution with ionic strength in the physiological range (0.2 M KCl, 20 mM imidazole, pH 7.2-7.4) (SMT(KCl) aggregates). Such aggregation in vivo, which may play a pathological or functional role, is not excluded. In view of the fact that some pathological amyloids can activate the classical and alternative pathways of complement system, we investigated the binding of complement component C1q and C3b to smooth muscle titin amyloid aggregates. The binding of С1q and C3b to SMT aggregates was not observed with ELISA assay. Since SMT aggregates do not activate the complement system, they are hardly implicated in the inflammatory process caused by muscle damage in amyloidoses.Abbreviations: SMT: smooth muscle titin; SMT(KCl) aggregates: SMT aggregates in solution containing 0.2 M KCl, 10 mM imidazole, pH 7.0; SMT(Gly) aggregates: SMT aggregates in solution containing 0.15 M glycine-KOH, pH 7.2-7.4; MAC: membrane attack complex; DLS: dynamic light scattering; NHS: Normal Human Serum.

—Changes in the expression of the titin gene and alternative splicing of titin pre-mRNA from exon 50 to exon 111 in rat soleus muscle were analyzed following 3-day functional unloading (the HS group). Using real-time RT-PCR, it was found that the expression level of the titin gene in the rat soleus muscle from the HS group was higher by 1.81 times (p ≤ 0.01, n = 6) than the control level (n = 7). It was shown that all studied exons of titin mRNA are present in rats soleus muscle from the two groups. Our results demonstrate that 3-day functional unloading is accompanied by an increase in the expression of the titin gene in rat soleus without changes in alternative splicing from exon 50 to exon 111.

—The hypothesis that formed the basis of this work has been made on our studies that have shown that giant multi-domain muscle proteins of the titin family (titin isoforms and myosin-binding protein C paralogs) form amyloid aggregates in vitro. We have identified a number of features of the amyloid aggregation of the above-mentioned proteins using a number of methods, including X-ray diffraction: (1) the ability to aggregate under reduced ionic strength at pH 7.0; (2) a high aggregation rate; (3) the formation of aggregates that have a quaternary structure like the cross-β structure, which is characteristic of many amyloids; (4) the absence of changes in the secondary structure in the formation of amyloid-like aggregates; and (5) the partial reversibility of amyloid aggregation. In this paper, we raise the following questions. Is amyloid aggregation of these proteins possible in vivo? If so, what could be the functional significance of such aggregation in a muscle cell? We believe that amyloid-like structures can be formed temporarily upon the interaction of individual molecules of multi-domain muscle proteins in the process of muscle contraction in the sarcomere. Such changes would certainly be accompanied an increase in the stiffness of the sarcomeres and the muscle in general, that can play a positive role in preventing harmful effects on the muscle under extreme physical exercise.

Alcoholic myopathy is characterized by the reduction in cross-sectional area (CSA) of muscle fibers and impaired anabolic signaling. The goal of the current study was to investigate the causes and compare the changes in CSA and fiber type composition with the modifications of anabolic and catabolic signaling pathways at the early stages of chronic alcohol consumption in women. Skeletal muscle samples from 5 female patients with alcohol abuse (AL; 43 ± 5 yr old; alcohol abuse duration 5,6 ± 0,6 yr) were compared with the muscle from the control group of 8 healthy women (C; 35 ± 4 yr old). The average daily dose of alcohol consumption was 110 ± 10 ml of pure ethanol. In women patients, a significant decrease in CSA of type I and II muscle fibers, titin and nebulin content, plasma IGF-1 level and total IRS-1, p-Akt and p-4E-BP1 in vastus lateralis was found in comparison with the control group. The p-AMPK level was found to be increased versus the control group. In women patients with chronic alcoholic myopathy 1) both fast and slow muscle fibers are subjected to atrophy; 2) impairments in IGF-I-dependent signaling and pathways controlling translation initiation (AMPK/mTOR/4E-BP1), but not translation elongation, are observed; 3) the level of calpain-1 and ubiquitinated proteins increases, unlike E3 ligases content.

Objective : to assess the structural and functional state of skeletal muscles in the hip, as well as changes in the content of the sarcomere cytoskeleton proteins titin and nebulin and their phosphorylation level in patients with chronic alcohol intoxication. Patients and methods . Thirteen patients (4 men and 9 women; mean age, 38.9±9.9 years) with chronic alcohol intoxication were examined. The average duration of regular consumption of alcoholic beverages was 7.6±3.7 years. The mean amount of alcohol consumed per week was 48.2±13.1 units. A control group included 10 healthy volunteers matched for age and sex. A neurological examination was performed according to the generally accepted scheme. Laboratory tests involved blood biochemical analysis estimating the levels of liver enzymes, creatine phosphokinase (CPK), and insulin-like growth factor-1 (IGF-1). All the patients underwent hip magnetic resonance imaging (MRI), followed by assessment of the degree of muscle tissue damage and by determination of the volume of anterior and posterior thigh muscles. The content of titin and nebulin and their phosphorylation level were determined in the muscle tissue samples obtained by an open biopsy from the lateral head of the quadriceps femoris muscle. Results and discussion . Four (30.8%) patients were found to have proximal leg muscle weakness; the degree of paresis was the same in the anterior and posterior thigh muscles. There was a significant increase in the plasma level of liver enzymes; the CPK level remained within the reference values; there was a tendency towards lower IGF-1 levels. Analysis of MRI data showed that 7 (53.8%) patients had fatty degeneration in the thigh muscles. Quantitative evaluation ascertained a significant symmetrical decrease in the volume of anterior thigh muscles and a tendency towards a symmetrical reduction in that in the posterior thigh muscle compared to the control. Analysis of the content of titin and nebulin in the lateral head of the quadriceps femoris muscle revealed a significant decrease in the percentage of nebulin (81.1%; p < 0.01) and intact titin-1 (T1) isoforms (83.6%; p < 0.01). The percentage of proteolytic titin-2 (T2) fragments in the muscle of patients did not differ significantly from that in the control group. Estimating the phosphorylation level of the structural muscle proteins showed no significant differences when compared to the control. Conclusion . Anterior and posterior thigh muscle weakness should be considered as the main clinical manifestation of chronic alcoholic myopathy (CAM) in the absence of biochemical and neurophysiological markers of the disease. Lower extremity muscle MRI that can reveal a lower muscle volume concurrent with fatty degeneration is a non-invasive informative diagnostic technique for CAM. The pathogenesis of skeletal muscle atrophy in chronic alcohol intoxication involves the sarcomere structural proteins titin and nebulin, which regulate the interaction of the major contractile proteins actin and myosin, and whole muscle contraction.

Simple Summary This review presents the beginning of the history of toxic properties of amyloids, especially on Aβ amyloids. We discuss anti-amyloid therapy and its problems and write about new views on amyloids that can play positive roles in different organisms including human. Abstract Proteins can perform their specific function due to their molecular structure. Partial or complete unfolding of the polypeptide chain may lead to the misfolding and aggregation of proteins in turn, resulting in the formation of different structures such as amyloid aggregates. Amyloids are rigid protein aggregates with the cross-β structure, resistant to most solvents and proteases. Because of their resistance to proteolysis, amyloid aggregates formed in the organism accumulate in tissues, promoting the development of various diseases called amyloidosis, for instance Alzheimer’s diseases (AD). According to the main hypothesis, it is considered that the cause of AD is the formation and accumulation of amyloid plaques of Aβ. That is why Aβ-amyloid is the most studied representative of amyloids. Therefore, in this review, special attention is paid to the history of Aβ-amyloid toxicity. We note the main problems with anti-amyloid therapy and write about new views on amyloids that can play positive roles in the different organisms including humans.

Various amyloid aggregates, in particular, aggregates of amyloid β-proteins, demonstrate in vitro and in vivo cytotoxic effects associated with impairment of cell adhesion. We investigated the effect of amyloid aggregates of smooth-muscle titin on smooth-muscle-cell cultures. The aggregates were shown to impair cell adhesion, which was accompanied by disorganization of the actin cytoskeleton, formation of filopodia, lamellipodia, and stress fibers. Cells died after a 72-h contact with the amyloid aggregates. To understand the causes of impairment, we studied the effect of the microtopology of a titin-amyloid-aggregate-coated surface on fibroblast adhesion by atomic force microscopy. The calculated surface roughness values varied from 2.7 to 4.9 nm, which can be a cause of highly antiadhesive properties of this surface. As all amyloids have the similar structure and properties, it is quite likely that the antiadhesive effect is also intrinsic to amyloid aggregates of other proteins. These results are important for understanding the mechanisms of the negative effect of amyloids on cell adhesion.

This work investigated in vitro aggregation and amyloid properties of skeletal myosin binding protein-C (sMyBP-C) interacting in vivo with proteins of thick and thin filaments in the sarcomeric A-disc. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) found a rapid (5&ndash, 10 min) formation of large (&gt, 2 &mu, m) aggregates. sMyBP-C oligomers formed both at the initial 5&ndash, 10 min and after 16 h of aggregation. Small angle X-ray scattering (SAXS) and DLS revealed sMyBP-C oligomers to consist of 7&ndash, 10 monomers. TEM and atomic force microscopy (AFM) showed sMyBP-C to form amorphous aggregates (and, to a lesser degree, fibrillar structures) exhibiting no toxicity on cell culture. X-ray diffraction of sMyBP-C aggregates registered reflections attributed to a cross-&beta, quaternary structure. Circular dichroism (CD) showed the formation of the amyloid-like structure to occur without changes in the sMyBP-C secondary structure. The obtained results indicating a high in vitro aggregability of sMyBP-C are, apparently, a consequence of structural features of the domain organization of proteins of this family. Formation of pathological amyloid or amyloid-like sMyBP-C aggregates in vivo is little probable due to amino-acid sequence low identity (&lt, 26%), alternating ordered/disordered regions in the protein molecule, and S&ndash, S bonds providing for general stability.

To date, some scientific evidence (limited proteolysis, mass spectrometry analysis, electron microscopy (EM)) has accumulated, which indicates that the generally accepted model of double-stranded of filamentous actin (F-actin) organization in eukaryotic cells is not the only one. This entails an ambiguous understanding of many of the key cellular processes in which F-actin is involved. For a detailed understanding of the mechanism of F-actin assembly and actin interaction with its partners, it is necessary to take into account the polymorphism of the structural organization of F-actin at the molecular level. Using electron microscopy, limited proteolysis, mass spectrometry, X-ray diffraction, and structural modeling we demonstrated that F-actin presented in the EM images has no double-stranded organization, the regions of protease resistance are accessible for action of proteases in F-actin models. Based on all data, a new spatial model of filamentous actin is proposed, and the F-actin polymorphism is discussed.

Molecular mechanisms underlying muscle-mass retention during hibernation have been extensively discussed in recent years. This work tested the assumption that protein synthesis hyperactivation during interbout arousal of the long-tailed ground squirrel Urocitellus undulatus should be accompanied by increased calpain-1 activity in striated muscles. Calpain-1 is known to be autolysed and activated in parallel. Western blotting detected increased amounts of autolysed calpain-1 fragments in the heart (1.54-fold, p p p p

This work studied the changes in the levels of the main proteins of the calpain system (μ-calpain, Ca^(2+)-dependent protease, and fragments of its autolysis, inhibitor calpastatin) and μ-calpain substrates (giant proteins of the sarcomere cytoskeleton, titin and nebulin) in skeletal muscle (m. gastrocnemius, m. soleus, m. longissimus dorsi) of rats alcoholized for three months by different methods using agar containing 30% ethanol and nutrient-balanced liquid feed containing 5% ethanol using gel electrophoresis methods under denaturing conditions and immunoblotting. No decrease in the muscle mass/body weight ratio, indicating the development of atrophy, no increase in autolysis of μ-calpain, indicating an increase in the activity of this enzyme, no changes in the content of intact titin (T1), nebulin, μ-calpain and calpastatin, as well as the total calpain activity measured using Calpain Activity Assay Kit were detected in alcoholized rats of both groups. No changes in the total level of titin phosphorylation in the rat muscles of alcoholized groups were detected using Pro-Q Diamond fluorescent dye for phosphate groups of proteins. No statistically significant differences in the content of titin and nebulin mRNA in skeletal muscles of control rats and rats alcoholized using agar were detected. In rats, alcoholized by the method of liquid feed, the levels of titin and nebulin mRNA were increased 1.5-2.5 times possibly due to a higher fat content in such a diet. The presented data may be useful for choosing a chronic alcoholization model for animals.

—Changes in the fatty acid levels in the cardiac and gastrocnemius muscles of rats that were chronically alcoholized for 3 and 6 months were studied using two methods of alcoholization: 30% ethanol-containing agar (method I) and a 5% ethanol-containing liquid diet with a balanced nutritional status (method II). In the control group, the fatty acid level in the cardiac muscle was considerably higher than that in the gastrocnemius muscle. In the animals that were alcoholized over a 3-month period using method I, a considerable increase in the levels of myristic, pentadecanoic, palmitic, stearic, and dihomo-γ-linolenic acids and the total amount of fatty acids and a decrease in ω-3 docosapentaenoic acid level were found in the cardiac muscle. After a 6-month period, during which rats were alcoholized using method I, an increase in the levels of palmitoleic and ω-6 docosapentaenoic acids and a decrease in the levels of stearic, eicosadienoic, and arachidonic acids were found. The amount of ω-3 docosapentaenoic acid in the myocardium, compared to that observed after a 3-month period during which rats were alcoholized, remained reduced compared to the control. In the gastrocnemius muscle of rats alcoholized for 3 months using method I, the amounts of myristic, vaccenic, dihomo-γ-linolenic, and ω-6 docosapentaenoic acid increased. Simultaneously, there was a tendency for the total amount of saturated, monounsaturated, and ω-6 polyunsaturated fatty acids and the total amount of fatty acids to rise. After a 6-month alcoholization, a decrease in the levels of myristic, oleic, linoleic, α- and γ-linolenic, and eicosadienoic acids, as well as the total amount of saturated, ω-6 polyunsaturated fatty acids and the total amount of all fatty acids was found. When animals were alcoholized over a 3-month period using method II, a significant increase in the amount of dihomo-γ-linolenic acid was detected in the cardiac and gastrocnemius muscles. The role of these changes in the muscle pathologies is discussed.

The scientific interest to the structural and functional properties of actin is determined by its abundance in cells. Being an important component of the cytoskeleton, actin is involved in many protein-protein interactions. Using crystal structures and molecular models, we have mapped the amino acid residues that are involved in these interactions and form the ATP-binding site of the actin monomer. Moreover, using mass spectrometry and high-performance liquid chromatography methods, we have discovered the regions of the amino acid sequence of actin that form the core of the actin fibril. According to the bioinformatic analysis, these regions are amyloidogenic and are located in the C-terminal region and in the hinge between the first and third subdomains. The data obtained are applicable to chordate actin, because multiple alignment revealed highly conserved amino acid sequences. In turn, the comparison of the chordate actin with the bacterial homologs showed the presence of numerous amino acid substitutions and insertions.

BACKGROUND Proteolysis can proceed via several distinct pathways such as the lysosomal, calcium-dependent, and ubiquitin-proteasome-dependent pathways. Calpains are the main proteases that cleave a large variety of proteins, including the giant sarcomeric proteins, titin and nebulin. Chronic ethanol feeding for 6 weeks did not affect the activities of μ-calpain and m-calpain in the m. gastrocnemius. In our research, changes in μ-calpain activity were studied in the m. gastrocnemius and m. soleus of chronically alcohol-fed rats after 6 months of alcohol intake. METHODS SDS-PAGE analysis was applied to detect changes in titin and nebulin contents. Titin phosphorylation analysis was performed using the fluorescent dye Pro-Q Diamond. Western blotting was used to determine μ-calpain autolysis as well as μ-calpain and calpastatin contents. The titin and nebulin mRNA levels were assessed by real-time PCR. RESULTS The amounts of the autolysed isoform (78 kDa) of full-length μ-calpain (80 kDa) increased in the m. gastrocnemius and m. soleus of alcohol-fed rats. The calpastatin content increased in m. gastrocnemius. Decreased intact titin-1 (T1) and increased T2-proteolytic fragment contents were found in the m. gastrocnemius and m. soleus of the alcohol-fed rats. The nebulin content decreased in the rat gastrocnemius muscle of the alcohol-fed group. The phosphorylation levels of T1 and T2 were increased in the m. gastrocnemius and m. soleus, and decreased titin and nebulin mRNA levels were observed in the m. gastrocnemius. The nebulin mRNA level was increased in the soleus muscle of the alcohol-fed rats. CONCLUSIONS In summary, our data suggest that prolonged chronic alcohol consumption for 6 months resulted in increased autolysis of μ-calpain in rat skeletal muscles. These changes were accompanied by reduced titin and nebulin contents, titin hyperphosphorylation, and development of hindlimb muscle atrophy in the alcohol-fed rats.

Almost 40 years has passed since the discovery of giant elastic protein titin (also known as connectin) of striated and smooth muscles using gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a major technique for studying the isoform composition and content of titin. This review provides historical insights into the technical aspects of the electrophoresis methods used to identify titin and its isoforms. We particularly focus on the nuances of the technique that improve the preservation of its primary structure so that its high molecular weight isoforms can be visualized.

The giant muscle protein, titin, is the third most abundant protein in muscle (after myosin and actin). It was shown previously that smooth muscle titin (SMT) with a molecular mass of 500 kDa can form in vitro amorphous amyloid aggregates in two conditions: in solution of low ionic strength (0.15 M Glycine-KOH, pH 7.0) (SMT(Gly) aggregates) and in solution with ionic strength in the physiological range (0.2 M KCl, 20 mM imidazole, pH 7.2-7.4) (SMT(KCl) aggregates). Such aggregation in vivo, which may play a pathological or functional role, is not excluded. In view of the fact that some pathological amyloids can activate the classical and alternative pathways of complement system, we investigated the binding of complement component C1q and C3b to smooth muscle titin amyloid aggregates. The binding of С1q and C3b to SMT aggregates was not observed with ELISA assay. Since SMT aggregates do not activate the complement system, they are hardly implicated in the inflammatory process caused by muscle damage in amyloidoses.

We studied the effect of histone deacetylase 1 (HDAC1) inhibition on titin content and expression of TTN gene in rat m. soleus after 3-day gravitational unloading. Male Wistar rats weighing 210±10 g were randomly divided into 3 groups: control, 3-day hindlimb suspension, and 3-day hindlimb suspension and injection of HDAC1 inhibitor CI-994 (1 mg/kg/day). In hindlimb-suspended rats, the muscle weight/animal body weight ratio was reduced by 13.8% (p

Seasonal changes in proteolytic activity and content of calpains in striated muscles of the longtailed ground squirrel Spermophilus undulatus were studied by casein zymography and Western blotting analysis. The results testify to hyperactivation of calpain proteases in the skeletal muscles of awakened animals during the "winter" activity. The observed changes are discussed in the context of adaptation of skeletal muscles of long-tailed ground squirrels to hibernation.

Staining with Congo Red (CR) is a qualitative method used for the identification of amyloids in vitro and in tissue sections. However, the drawbacks and artefacts obtained when using this dye can be found both in vitro and in vivo. Analysis of scientific data from previous studies shows that CR staining alone is not sufficient for confirmation of the amyloid nature of protein aggregates in vitro or for diagnosis of amyloidosis in tissue sections. In the present paper, we describe the characteristics and limitations of other methods used for amyloid studies. Our historical review on the use of CR staining for amyloid studies may provide insight into the pitfalls and caveats related to this technique for researchers considering using this dye.

This review considers data on structural and functional features of titin, on the role of this protein in determination of mechanical properties of sarcomeres, and on specific features of regulation of the stiffness and elasticity of its molecules, amyloid aggregation of this protein in vitro, and possibilities of formation of intramolecular amyloid structure in vivo. Molecular mechanisms are described of protection of titin against aggregation in muscle cells. Based on the data analysis, it is supposed that titin and the formed by it elastic filaments have features of amyloid.

Seasonal changes in the isoform composition of thick and thin filament proteins (titin, myosin heavy chains (MyHCs), nebulin), as well as in the phosphorylation level of titin in striated muscles of brown bear (Ursus arctos) and hibernating Himalayan black bear (Ursus thibetanus ussuricus) were studied. We found that the changes that lead to skeletal muscle atrophy in bears during hibernation are not accompanied by a decrease in the content of nebulin and intact titin-1 (T1) isoforms. However, a decrease (2.1-3.4-fold) in the content of T2 fragments of titin was observed in bear skeletal muscles (m. gastrocnemius, m. longissimus dorsi, m. biceps) during hibernation. The content of the stiffer N2B titin isoform was observed to increase relative to the content of its more compliant N2BA isoform in the left ventricles of hibernating bears. At the same time, in spite of the absence of decrease in the total content of T1 in the myocardium of hibernating brown bear, the content of T2 fragments decreased ~1.6-fold. The level of titin phosphorylation only slightly increased in the cardiac muscle of hibernating brown bear. In the skeletal muscles of brown bear, the level of titin phosphorylation did not vary between seasons. However, changes in the composition of MyHCs aimed at increasing the content of slow (I) and decreasing the content of fast (IIa) isoforms of this protein during hibernation of brown bear were detected. Content of MyHCs I and IIa in the skeletal muscles of hibernating Himalayan black bear corresponded to that in the skeletal muscles of hibernating brown bear.

A comparative study of amyloid properties of the aggregates of smooth muscle titin (SMT) from chicken gizzard was carried out. These aggregates were formed in two solutions: 0.15 M glycine-KOH, pH 7.2–7.4 (SMT(Gly)) and 0.2 M KCl, 10 mM imidazole, pH 7.0 (SMT(KCl)). Electron microscopy data showed that SMT aggregates has an amorphous structure in both cases. The results of atomic-force microscopy demonstrated slight differences in morphology in two types of aggregates. The SMT(Gly) aggregates were represented as branching chains, composed of spherical aggregates approximately 300–500 nm in diameter and up to 35 nm in height. The SMT(KCl) aggregates formed sponge-like structures with strands of 8–10 nm in height. Structural analysis of SMT aggregates by X-ray diffraction revealed the presence of cross-β-sheet structure in the samples under study. In the presence of SMT(Gly) aggregates, thioflavine T fluorescence intensity was higher (~3-fold times) compared with that in the presence of SMT(KCl) aggregates. Congo red-stained SMT(Gly) aggregates had yellow to apple-green birefringence under polarized light, which was not observed for SMT(KCl) aggregates. Dynamic light scattering data showed the similar rate of aggregation for both types of aggregates, though SMT(KCl) aggregates were able to partially disaggregate under increased ionic strength of the solution. The ability of SMT to aggregation followed by disaggregation may be functionally significant in the cell.

Enzymatic activity of Ca2+-dependent calpain proteases as well as the content and gene expression of μ-calpain (activated by micromolar calcium ion concentrations), calpastatin (inhibitor of calpains), and titin (substrate for calpains) were investigated in cardiac muscles of rats subjected to chronic alcoholization for 3 and 6 months. There was no increase in the “heart weight/body weight” parameter indicating development of heart hypertrophy in the alcoholized rats, while a decreasing trend was observed for this parameter in the rats after 6-month modeling of alcoholic cardiomyopathy, which indicated development of atrophic changes in the myocardium. Fluorometric measurements conducted using the Calpain Activity Assay Kit did not reveal any changes in total calpain activity in protein extracts of cardiac muscles of the rats alcoholized for 3 and 6 months. Western blot analysis did not show reliable changes in the contents of μ-calpain and calpastatin, and SDS-PAGE did not reveal any decrease in the titin content in the myocardium of rats after the chronic alcohol intoxication. Autolysis of μ-calpain was also not verified, which could indicate that proteolytic activity of this enzyme in myocardium of chronically alcoholized rats is not enhanced. Using Pro-Q Diamond staining, changes in phosphorylation level of titin were not detected in cardiac muscle of rats after chronic alcoholization during three and six months. A decrease in μ-calpain and calpastatin mRNA content (~1.3-fold, p ≤ 0.01 and ~1.9-fold, p ≤ 0.01, respectively) in the myocardium of rats alcoholized for 3 months and decrease in calpastatin mRNA (~1.4-fold, p ≤ 0.01) in animals alcoholized for 6 months was demonstrated using real-time PCR. These results indicate negative effect of chronic alcohol intoxication on expression of the abovementioned genes.

Using a spectrophotometric method changes occurring in solution containing brain Aβ(1-42)-peptide, fullerene C60, and polyvinylpyrrolidone were analyzed. Using the Bent-French method relative binding constants of fullerene C60 with Aβ(1-42)-peptide and polyvinylpyrrolidone with Aβ(1-42)- peptide were determined. These data suggest that Aβ(1-42)-peptide interacting with the C60 fullerene-polyvinylpyrrolidone complex partially displaces polyvinylpyrrolidone and generates a new three molecular compound.

Anti-amyloid activity, aggregation behaviour, cytotoxicity and acute toxicity were investigated for three water-soluble fullerene derivatives with different types of solubilizing addends. All investigated compounds showed a strong anti-amyloid effect in vitrocaused by interaction of the water-soluble fullerene derivatives with the Ab(1-42)-peptide and followed by destruction of the amyloid fibrils. Notably, all of the studied fullerene derivatives showed very low cytotoxicity and low acute toxicity in mice (most promising compound 3 was more than four times less toxic than aspirin). Strong anti-amyloid effect of the fullerene derivatives together with low toxicity reveals high potential of these compounds as drug candidates for treatment of neurodegenerative diseases.

From our earlier experiments on the study of changes in titin content and the level of its phosphorylation in skeletal muscles, atrophied during space flight, hibernation, and also because of the development of alcohol-induced lesions it has been suggested that an increase in the degree of titin phosphorylation results in increased proteolytic degradation of this protein, that contributes to the development of skeletal muscle atrophy.

Amyloids are insoluble fibrous protein aggregates, and their accumulation is associated with amyloidosis and many neurodegenerative diseases, including Alzheimer's disease. In the present study, we report that smooth muscle titin (SMT; 500 kDa) from chicken gizzard forms amyloid aggregates in vitro. This conclusion is supported by EM data, fluorescence analysis using thioflavin T (ThT), Congo red (CR) spectroscopy and X-ray diffraction. Our dynamic light scattering (DLS) data show that titin forms in vitro amyloid aggregates with a hydrodynamic radius (Rh) of approximately 700–4500 nm. The initial titin aggregates with Rh approximately 700 nm were observed beyond first 20 min its aggregation that shows a high rate of amyloid formation by this protein. We also showed using confocal microscopy the cytotoxic effect of SMT amyloid aggregates on smooth muscle cells from bovine aorta. This effect involves the disorganization of the actin cytoskeleton and result is cell damage. Cumulatively, our results indicate that titin may be involved in generation of amyloidosis in smooth muscles.

This review summarizes results of our studies on titin isoform composition in vertebrate striated muscles under normal conditions, during hibernation, real and simulated microgravity, and under pathological conditions (stiff-person syndrome, post-apoplectic spasticity, dilated cardiomyopathy, cardiac hypertrophy). Experimental evidence for the existence in mammalian striated muscles of higher molecular weight isoforms of titin (NT-isoforms) in addition to the known N2A-, N2BA-, and N2B-titin isoforms was obtained. Comparative studies of changes in titin isoform composition and structure-functional properties of human and animal striated muscles during adaptive and pathological processes led to a conclusion about the key role of NT-isoforms of titin in maintenance of sarcomere structure and contractile function of these muscles.

Seasonal changes of the isoform composition of myosin heavy chains in skeletal muscles (m. triceps, m. longissimus dorsi, m. soleus, m. gastrocnemius, m. vastus lateralis) of hibernating ground squirrels Spermophilus undulatus were studied. Functional properties of myosin (the actin-activated ATPase activity and its Ca2+-sensitivity in vitro) were also examined. It was observed that the content of slow myosin heavy chain I isoform increased and the content of fast IIx/d isoform decreased in muscles of torpid ground squirrels and animals which are active in autumn and winter. In muscles of these animals the content of N2A-titin isoform decreased although the relative content of NT-titin isoform, observed in striated muscles of mammals in our previous experimental works, increased. Actin-activated ATPase activity and Ca2+-sensitivity of myosin isolated from skeletal muscles of torpid and interbout ground squirrels were found to reduce. The changes observed are discussed in the context of adaptation of skeletal muscles of ground squirrels to hibernation conditions.

Viability, histology and ultrastructure of normal cells and cells of different degrees of malignancy after interaction with dopamine as well as the ability of these cells and isolated G-actin in model experiments to stain by Falck technique were studied. It is shown that dopamine, virtually having no effect on the viability of the “normal” non-tumorigenic transformed cells, noticeably reduces cell viability of slightly tumorigenic cells, causes a significant reduction in viability of attachable cancerous cells and a very significant decrease in cell viability of cancerous cells growing in suspension. The intensity of fluorescence of the cytosol in cells treated with dopamine, has been very high and varied in different cultures, and that of isolated actin directly depended on its concentration. Common to all cell morphological feature of damage from the action of dopamine and the putative substrate of fluorescence was actodopamine filaments network strands (identified on the structure and size), which appears in the cytosol loci, where they were absent in control. The data show that dopamine can be used as an oncotherapeutic remedy and diagnostic tool interacting with G-actin as a cellular target.

Changes of titin and myosin heavy chain isoform composition in skeletal muscles (m. soleus, m. gastrocnemius, m. tibialis anterior, m. psoas major) in Mongolian Gerbil (Meriones unguiculatus) were investigated after 12-day spaceflight on board of Russian space vehicle “Foton-M3.” In m. psoas and m. soleus in the gerbils from “Flight” group the expected increase in the content of fast myosin heavy chain isoforms (IIxd and IIa, respectively) were observed. No significant differences were found in the content of IIxd and IIa isoforms of myosin heavy chain in m. tibialis anterior in the gerbils from control group as compared to that in “Flight” group. An unexpected increase in the content of slow myosin heavy chain I isoform and a decrease in the content of fast IIx/d isoform in m. gastrocnemius of the gerbils from “Flight” group were observed. In skeletal muscles of the gerbils from “Flight” group the relative content of titin N2A-isoform was reduced (by 1.2–1.7 times), although the content of its NT-isoform, which was revealed in striated muscles of mammals in our experiments earlier, remained the same. When the content of titin N2A-isoform was decreased, no predictable abnormalities in sarcomeric structure and contractile ability of skeletal muscles in the gerbils from “Flight” group were found. An assumption on the leading role of titin NT-isoform in maintenance of structural and functional properties of striated muscles of mammals was made.

The data of the study on Ca2+ sensitivity of ATPase activity of myosin from vertebrate striated muscles in the presence of actin and the conditions of its manifestation and disappearance are presented. The role of Ca2+ sensitivity of actin-activated myosin ATPase in the regulation of contraction of vertebrate striated muscles is discussed.

Changes in the expression of titin N2B and N2BA isoforms in the left ventricle of the heart of spontaneously hypertensive rats during the development of hypertrophy have been analyzed by the methods of real-time polymerase chain reaction and SDS gel electrophoresis. It was shown that, in early terms of development of hypertrophy (15-week-old rats), an increase in the expression of mRNA of the titin gene and a decrease in the content of the protein itself occur. At a later stage of development (26-week-old rats), a decrease in the expression of titin at the level of both mRNA and protein was observed. The results obtained can be used in the development of methods for diagnosing the development of myocardial hypertrophy.

An electophoretic study of changes in the content of intact titin isoforms, N2B-, N2BA-, N2A-titins and T2 in skeletal and cardiac muscles of ground squirrel (Spermophillus undulatus) is made in different periods: summer activity, autumnal activity, hibernation, arousal, and winter activity. In atria and ventricles of ground squirrels in the period of autumnal activity an increase (by ∼1.5 times) in the N2BA to N2B ratio was observed, in comparison with that in cardiac muscle in summer activity. During hibernation, the decrease in the relative content of N2B-, N2BA-titins and T2 in cardiac muscle as well as of N2A-titin and T2 in skeletal muscles was determined against the background of preservation of the relative amount of intact titin isoforms. At waking of ground squirrels and in a short period of winter activity, a rapid restoration of the content of N2B-, N2BA-, N2A-titisns and T2 in muscles was observed. In the myocardium of hibernating, waking ground squirrels and of those during winter activity the increased N2BA to N2B ratio was retained. The changes in the titin content are discussed in the aspect of adaptation of ground squirrels to hibernation.

An electophoretic study of changes in composition of titin isoforms in human and rat skeletal and cardiac muscles is carried out. A more considerable decrease in the content of intact titin isoforms was observed than in the content of N2A-titin in the dorsal muscle of patients with the “stiff-person syndrome” and in m. soleus of humans and rats during development of “muscle hypogravity syndrome” and than in the content of N2BA- and N2B-titins in hypertrophic heart of spontaneously hypertensive rats. The relation between reduction of titin content in m. soleus and the increase of time the rats were in conditions of simulated microgravity is revealed. On electrophoregrams of left ventricle myocardium of patients with terminal stage of dilated cardiomyopathy the intact titin and N2BA-titin were absent and a considerable decrease in the content of N2B-titin was observed. This could be the consequence of the terminal stage of pathology. It follows that development of the diseases is accompanied by a greater destruction of intact titin than of its other forms which may be important for diagnostics of pathological processes.

The contractile properties of the postural rat soleus muscle at the early stage of the gravitational unloading (3-day rat hindlimb suspension) have been studied using different modes of muscle contraction (twitch and tetanic contraction of the isolated muscle, Ca-induced contraction of isolated skinned fibers). A significant enhancement of the twitch maximal tension of unloaded muscles without changes in time-dependent characteristics was observed, although the half-relaxation time tended to increase. The fiber diameter did not change (42.37 +/- 0.76 vs 43.43 +/- 1.15 microm in controls). The Ca-induced maximal isometric tension in unloaded soleus was significantly decreased (32.1 +/- 1.05 vs 37.6 +/- 1.52 mg in controls, p < 0.05). The maximal specific tension was respectively decreased (23.14 +/- 0.77 vs 27.6 +/- 2.36 kN/m in controls). The pCa50 in unloaded muscle decreased from 6.05 +/- 0.02 in controls to 5.97 +/- 0.02 (p < 0.05), indicating the loss of myofibrillar calcium sensitivity. The analysis with the calcium probe Fluo-4AM demonstrated that the intracellular [Ca2+] was sufficiently increased after hindlimb suspension. At the same time, the relative content of titin and nebulin did not change.